Toxoplama gondii is an obligate intracellular parasite and the causative agent of Toxoplasmosis which, if left untreated, can cause life-threatening disease in immunocompromised individuals and neonates. Pathogenesis of the disease relies on the parasites ability to survive and replicate within a specialized organelle in the host, the parasitophorous vacuole, (PV). Proteins released from secretory vesicles, the dense granules (DGs), have diverse functions which are critical for intracellular survival including modification f PV structure and metabolic activity, regulation of host cell cycle and modification of host gene expression. How the dense granules, small 200nm membrane enclosed vesicles, are transported within the parasite and their contents released into the host cell are major outstanding questions. Understanding this essential process on a mechanistic level would lead to the identification of potential targets of anti-parasitic drug development. Research progress in this area has been hampered to date by the inability to image individual granules on their path to secretion in real-time, in live parasites. The goal of this R21 proposal is to develop the advanced imaging techniques and biological reagents to overcome this hurdle and to begin elucidating the molecular mechanisms underlying DG transport and secretion. I have already imaged dense granule motions in live parasites with high spatial (30nm) and temporal (10fps) resolution. My preliminary data shows that TgMyoF, an uncharacterized class XXVII myosin motor, is necessary for DG transport in T. gondii. I hypothesize that DGs are transported processively by TgMyoF along actin tracks to their destination. Thus, in Aim 1 I will define TgMyoF's functional capacity to transport cargo in vitro and in parasites using cutting edge single molecule biophysical techniques. Then, in Aim 2 I will determine how TgMyoF-based transport facilitates DG secretion. Since DG transport occurs predominately at the parasite periphery I hypothesize that the DGs are transported until the granule encounters a specific site of accessibility that allow DGs to traverse the inner membrane complex (IMC) and reach the plasma membrane for secretion (IMC; a series of flattened vesicles that are located at the parasite periphery, which likely act as a barrier to secretion). To test this hypothesis DG secretion events will be imaged in both wildtype and TgMyoF deficient parasite lines to determine how loss of TgMyoF-based transport affects DG secretion. Collectively, these studies will provide fundamental new information about the process of DG transport and secretion.